Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

BACKGROUND: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). OBJECTIVE: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. METHODS: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. RESULTS: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. CONCLUSION: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

[ANTIINFLAMMATORY AND REGENERATIVE EFFECT OF PEPTIDE THERAPY IN THE MODEL OF OBSTRUCTIVE LUNG PATHOLOGY].

The effect of the tetrapeptide bronchogen on the structural and functional state of the bronchial epithelium and inflammatory activity in the lungs was studied in the chronic obstructive pulmonary disease (COPD) model, created in rats by a 60-day intermittent exposure to nitrogen dioxide. The cell composition and cytokine-enzyme profile of bronchoalveolar lavage fluid (BALF), the content of secretory immunoglobulin A and surfactant protein B in BALF were determined. Following the course of peptide treatment the decreased activity of neutrophilic inflammation with the normalization of cellular composition and profile of pro-inflammatory cytokines and enzymes in the bronchoalveolar space was observed. The structure of bronchial epithelium, disturbed during formation of COPD model, was restored and accompanied by restoration of its functional activity as evidenced by an increase of secretory immunoglobulin A (local immunity marker) and surfactant protein B, responsible for reducing the alveolar surface tension.

Interindividual variability in the expression of surfactant protein A and B in the human lung during development.

The surfactant complex, thanks to its multiple actions including decrease of surface- tension and antimicrobial activity, plays a fundamental role in newborn survival, lowering the risk of respiratory distress syndrome. The aim of this work was to determine if the synthesis of two surfactant proteins (SP), SPA and pro-SPB, shows some inter-individual variability during lung development in the intrauterine life. Immunoreactivity for SPA and pro-SPB was investigated in the lungs of  40 subjects, including 15 fetuses, ranging from 14 to 22 weeks of gestation, and 25 neonates, from 24 to 41 weeks. Lung samples were formalin fixed, paraffin-embedded and routinely processed. SPA and pro-SPB were detected utilizing commercial antibodies.  A semi-quantitative grading system (1 to 4) was applied, based on the number of reactive cells and the intensity of immunostaining. Surfactant protein immunostaining was found in  three compartments: bronchi, bronchioles and alveoli, starting from 14 weeks of gestation in the bronchial epithelium and from the 21st week in the alveolar spaces. Differences were found regarding SPA and pro-SPB expression in the vast majority of subjects: in some lungs, SPA was more expressed whereas in others pro-SPB showed an higher degree of immunoreactivity. The expression of both surfactant proteins was not strictly correlated with gestational age. Whereas the highest levels of reactivity were detected in at term neonates, on the other hand one case with grade 3 was detected at 22 weeks and one negative case for both proteins was observed at 31 weeks. Our data clearly show a marked inter-individual variability regarding the production of SPA and pro-SPB and suggest the existence of other epigenetic factors, acting during gestation, that might influence surfactant production and, consequently, the survival potential of  neonates at birth.

Comparative study of the effects of PM1-induced oxidative stress on autophagy and surfactant protein B and C expressions in lung alveolar type II epithelial MLE-12 cells.

BACKGROUND: There is a strong link between smaller air pollution particles and a range of serious health conditions. Thus, there is a need for understanding the impacts of airborne fine particulate matter (PM) with an aerodynamic diameter of <1µm (PM1) on lung alveolar epithelial cells. In the present study, mouse lung epithelial type II cell MLE-12 cells were used to examine the intracellular oxidative responses and the surfactant protein expressions after exposure to various concentrations of PM1 collected from an urban site and a steel-factory site (referred as uPM1 and sPM1 hereafter, respectively). METHODS: Physicochemical characterization of PM1 was performed by using scanning electron microscopy and transmission electron microscopy. Cytotoxicity and autophagy induced by PM1 were assessed by using comprehensive approaches after MLE-12 cells were exposed to different concentrations of PM1 for various times. Expression of surfactant proteins B and C in MLE-12 cells was determined by Western blotting. RESULTS: All of the tested PM1 induced cytotoxicity evidenced by significant decrease of cell viability and increase of lactate dehydrogenase (LDH) release in a time- and concentration-dependent manner in the exposed cells compared with the unexposed cells. A similar pattern of increase of intercellular reactive oxygen species (ROS) generation and decrease of superoxide dismutase (SOD) and catalase (CAT) activities was also observed. PM1-induced autophagy was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and increased levels of beclin1. Data from Western blotting showed significant decrease of surfactant protein B and C expressions. Relatively high concentrations of transition metals, including Fe, Cu and Mn, may be responsible for the higher toxicity of sPM1 compared with uPM1. Moreover, pretreatment with N-acetylcysteine (NAC) or Chelex (a metal chelating agent, which removes a large suite of metals from PM1) prevented the increase of PM1-inudced ROS generation and autophagy, and down-regulated the expression of surfactant proteins B and C. CONCLUSION: PM1, particularly PM1 with high concentrations of transition metals, such as Fe, Cu and Mn, induces oxidative damage and autophagy, as well as inhibits surfactant protein B and C expressions in lung alveolar type II epithelial cells. GENERAL SIGNIFICANCE: This study will help to understand the mechanism underlying the toxicological effects of PM1 in lung alveolar type II epithelial cells. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Respiratory adaptation and surfactant composition of unanesthetized male and female lambs differ for up to 8 h after preterm birth.

BACKGROUND: Male preterm infants are more likely to experience respiratory distress syndrome than females. Our objectives were to determine if sex-related differences in physiological adaptation after preterm birth increase with time after birth and if the use of continuous positive airway pressure (CPAP) reduces these differences. METHODS: Unanesthetized lambs (9F, 8M) were delivered at 0.90 of term. Blood gases, metabolites, and cardiovascular and respiratory parameters were monitored in spontaneously breathing lambs for 8 h. Supplemental oxygen was administered via a face mask at 4 cmH2O CPAP. At 8 h, lung compliance was determined, and bronchoalveolar lavage fluid (BALF) was analyzed for total protein and surfactant phospholipids. Surfactant protein (SP) gene expression and protein expression of SP-A and pro-SP-C were determined in lung tissue. RESULTS: For 8 h after delivery, males had significantly lower arterial pH and higher Paco2, and a greater percentage of males were dependent on supplemental oxygen than females. Inspiratory effort was greater and lung compliance was lower in male lambs. Total protein concentration in BALF, SP gene expression, and SP-A protein levels were not different between sexes; pro-SP-C was 24% lower in males. CONCLUSION: The use of CPAP did not eliminate the male disadvantage, which continues for up to 8 h after preterm birth.

Effects of oleic acid on SP-B expression and release in A549 cells.

OBJECTIVE: Pulmonary surfactant-associated protein B (SP-B), which is synthesized and secreted by alveolar epithelial type II cells, is crucial for normal functioning of pulmonary surfactant. Degeneration of pulmonary surfactant is the essential cause of acute lung injury (ALI). ALI is often studied in animal models using oleic acid, and the effects of oleic acid on pulmonary surfactant and SP-B are not clear. In this study, we examined the effects of oleic acid on the A549 cell line which resembles the alveolar epithelial type II cells. MATERIALS AND METHODS: A549 cells were exposed for 24 hours to 300, 400, 500 or 600 µM of oleic acid. Cell morphological changes were observed using an inverted microscope, and cell proliferation was quantified with the Cell Counting Kit-8. Extracellular SP-B levels were assessed by ELISA, whereas intracellular SP-B expression by Western blot. RESULTS: Oleic acid caused dose-dependent changes in cell morphology of A549 cells and decreased their proliferation. This was accompanied by release of SP-B into extracellular supernatants and corresponding decrease of intracellular levels of this protein. CONCLUSIONS: Oleic acid causes a dose-dependent injury to A549 cells, release of SP-B into extracellular compartment, and decrease of intracellular SP-B expression. Our findings provide mechanistic insights into animal modeling of ALI with oleic acid.

A preterm pig model of lung immaturity and spontaneous infant respiratory distress syndrome.

Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia remain the leading causes of preterm infant morbidity, mortality, and lifelong disability. Research to improve outcomes requires translational large animal models for RDS. Preterm pigs delivered by caesarian section at gestation days (GD) 98, 100, 102, and 104 were provided 24 h of neonatal intensive care, monitoring (pulse oximetry, blood gases, serum biomarkers, radiography), and nutritional support, with or without intubation and mechanical ventilation (MV; pressure control ventilation with volume guarantee). Spontaneous development of RDS and mortality without MV are inversely related with GD at delivery and correspond with inadequacy of tidal volume and gas exchange. GD 98 and 100 pigs have consolidated lungs, immature alveolar architecture, and minimal surfactant protein-B expression, and MV is essential at GD 98. Although GD 102 pigs had some alveoli lined by pneumocytes and surfactant was released in response to MV, blood gases and radiography revealed limited recruitment 1-2 h after delivery, and mortality at 24 h was 66% (35/53) with supplemental oxygen provided by a mask and 69% (9/13) with bubble continuous positive airway pressure (8-9 cmH2O). The lungs at GD 104 had higher densities of thin-walled alveoli that secreted surfactant, and MV was not essential. Between GD 98 and 102, preterm pigs have ventilation inadequacies and risks of RDS that mimic those of preterm infants born during the saccular phase of lung development, are compatible with standards of neonatal intensive care, and are alternative to fetal nonhuman primates and lambs.

Stage-dependent and locus-specific role of histone demethylase Jumonji D3 (JMJD3) in the embryonic stages of lung development.

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.

LPS-treated macrophage cytokines repress surfactant protein-B in lung epithelial cells.

In the mouse lung, Escherichia coli LPS can decrease surfactant protein-B (SFTPB) mRNA and protein concentrations. LPS also regulates the expression, synthesis, and concentrations of a variety of gene and metabolic products that inhibit SFTPB gene expression. The purpose of the present study was to determine whether LPS acts directly or indirectly on pulmonary epithelial cells to trigger signaling pathways that inhibit SFTPB expression, and whether the transcription factor CCAAT/enhancer binding protein (C/EBP)-ß (CEBPB) is a downstream inhibitory effector. To investigate the mechanism of SFTPB repression, the human pulmonary epithelial cell lines NCI-H441 (H441) and NCI-H820 (H820) and the mouse macrophage-like cell line RAW264.7 were treated with LPS. Whereas LPS did not decrease SFTPB transcripts in H441 or H820 cells, the conditioned medium of LPS-treated RAW264.7 cells decreased SFTPB transcripts in H441 and H820 cells, and inhibited SFTPB promoter activity in H441 cells. In the presence of neutralizing anti-tumor necrosis factor (TNF) antibodies, the conditioned medium of LPS-treated RAW264.7 cells did not inhibit SFTPB promoter activity. In H441 cells treated with recombinant TNF protein, SFTPB transcripts decreased, whereas CEBPB transcripts increased and the transient coexpression of CEBPB decreased SFTPB promoter activity. Further, CEBPB short, interfering RNA increased basal SFTPB transcripts and countered the decrease of SFTPB transcripts by TNF. Together, these findings suggest that macrophages participate in the repression of SFTPB expression by LPS, and that macrophage-released cytokines (including TNF) regulate the transcription factor CEBPB, which can function as a downstream transcriptional repressor of SFTPB gene expression in pulmonary epithelial cells.

Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4-Cre-mediated knockout of the tuberous sclerosis complex 1 gene.

Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1-mTORC1 may be critical for the synthesis of surfactant proteins A and B.

Synergistic effect of caffeine and glucocorticoids on expression of surfactant protein B (SP-B) mRNA.

Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis.

Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

Prenatal iodine deficiency results in structurally and functionally immature lungs in neonatal rats.

Maternal hypothyroidism affects postnatal lung structure. High prevalence of hypothyroxinemia (low T4, normal T3) in iodine-deficient pregnant women and associated risk for neuropsychological development along with high infant/neonatal mortality ascribed to respiratory distress prompted us to study the effects of maternal hypothyroxinemia on postnatal lung development. Female Sprague Dawley rats were given a low-iodine diet (LID) with 1% KClO(4) in drinking water for 10 days, to minimize thyroid hormone differences. Half of these rats were continued on iodine-deficient diet; ID (LID with 0.005% KClO(4)) for 3 mo, whereas the rest were switched to an iodine-sufficient diet; IS [LID + potassium iodide (10 µg iodine/20 g of diet + normal drinking water)]. Pups born to ID mothers were compared with age-matched pups from IS mothers at postnatal days 8 (P8) and 16 (P16) (n = 6-8/group). ID pups had normal circulating T3 but significantly low T4 levels (P < 0.05) and concomitantly approximately sixfold higher thyroid hormone receptor-ß mRNA in alveolar epithelium. Lung histology revealed larger and irregularly shaped alveoli in ID pups relative to controls. Lung function was assessed at P16 using a double-chambered plethysmograph and observed reduced tidal volume, peak inspiratory and expiratory flow, and dynamic lung compliance in ID pups compared with IS pups. Significant lowering of surfactant protein (SP)-B and SP-C mRNA and protein found in ID pups at P16. ID pups had 16-fold lower matrix metalloproteinase-9 mRNA levels in their alveolar epithelium. In addition, mRNA levels of thyroid transcription factor-1 and SP-D were significantly higher (3-fold) compared with IS pups. At P16, significantly lower levels of SP-B and SP-C found in ID pups may be responsible for immature lung development and reduced lung compliance. Our data suggest that maternal hypothyroxinemia may result in the development of immature lungs that, through respiratory distress, could contribute to the observed high infant mortality in ID neonates.

Neutral lipid trafficking regulates alveolar type II cell surfactant phospholipid and surfactant protein expression.

Adipocyte differentiation-related protein (ADRP) is a critically important protein that mediates lipid uptake, and is highly expressed in lung lipofibroblasts (LIFs). Triacylglycerol secreted from the pulmonary circulation and stored in lipid storage droplets is a robust hormonal-, growth factor-, and stretch-regulated precursor for surfactant phospholipid synthesis by alveolar type II epithelial (ATII) cells. A549 lung epithelial cells rapidly take up green fluorescent protein (GFP)-ADRP fusion protein-associated lipid droplets (LDs) in a dose-dependent manner. The LDs initially localize to the perinuclear region of the cell, followed by localization in the cytoplasm. Uptake of ADRP-LDs causes a time- and dose-dependent increase in surfactant protein-B (SP-B) expression. This mechanism can be inhibited by either actinomycin D or cycloheximide, indicating that ADRP-LDs induce newly synthesized SP-B. ADRP-LDs concomitantly stimulate saturated phosphatidylcholine (satPC) synthesis by A549 cells, which is inhibited by ADRP antibody, indicating that this is a receptor-mediated mechanism. Intravenous administration of GFP-ADRP LDs to adult rats results in dose-dependent increases in lung ADRP and SP-B expression. These data indicate that lipofibroblast-derived ADRP coordinates ATII cells' synthesis of the surfactant phospholipid-protein complex by stimulating both satPC and SP-B. The authors propose, therefore, that ADRP is the physiologic determinant for the elusive coordinated, stoichiometric synthesis of surfactant phospholipid and protein by pulmonary ATII cells.

Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells.

Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults.

Disaturated-phosphatidylcholine and surfactant protein-B turnover in human acute lung injury and in control patients.

BACKGROUND: Patients with adult respiratory distress syndrome (ARDS) and acute lung injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. OBJECTIVES: To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls). METHODS: 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. RESULTS: 1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively). CONCLUSIONS: 1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

Transcriptional regulation of SP-B gene expression by nitric oxide in H441 lung epithelial cells.

Surfactant protein B (SP-B) is essential for the surface tension-lowering function of pulmonary surfactant. Surfactant dysfunction and reduced SP-B levels are associated with elevated nitric oxide (NO) in inflammatory lung diseases, such as acute respiratory distress syndrome. We previously found that NO donors decreased SP-B expression in H441 and MLE-12 lung epithelial cells by reducing SP-B promoter activity. In this study, we determined the roles of DNA elements and interacting transcription factors necessary for NO inhibition of SP-B promoter activity in H441 cells. We found that the NO donor diethylenetriamine-nitric oxide adduct (DETA-NO) decreased SP-B promoter thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3 (HNF-3), and Sp1 binding activities but increased activator protein 1 (AP-1) binding activity. DETA-NO decreased TTF-1, but not Sp1, levels, suggesting that reduced TTF-1 expression contributes to reduced TTF-1 binding activity. Lack of effect on Sp1 levels suggested that DETA-NO inhibits Sp1 binding activity per se. Overexpression of Sp1, but not TTF-1, blocked DETA-NO inhibition of SP-B promoter activity. DETA-NO inhibited SP-B promoter induction by exogenous TTF-1 without altering TTF-1 levels. DETA-NO decreased TTF-1 mRNA levels and gene transcription rate, indicating that DETA-NO inhibits TTF-1 expression at the transcriptional level. We conclude that NO inhibits SP-B promoter by decreasing TTF-1, Sp1, and HNF-3 binding activities and increasing AP-1 binding activity. NO inhibits TTF-1 levels and activity to decrease SP-B expression. NO inhibition of SP-B expression could be a mechanism by which surfactant dysfunction occurs in inflammatory lung diseases.

Effect of inhaled budesonide on surfactant protein expression in asthmatic mice.

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response. Inhaled corticosteroids have been routinely used in the treatment of asthma, but neither its exact role nor its regulation on pulmonary surfactant is completely understood. The aim of this study was to determine the effect of budesonide on surfactant protein (SP) expression in asthmatic mice. Moreover, we investigated the function of transforming growth factor (TGF) beta signaling in the pulmonary surfactant system to identify a novel target for the treatment of asthma. Mice were sensitized and challenged by ovalbumin (OVA) to establish a murine model of asthma. To assess the effect of budesonide on asthmatic mice, animals were treated with aerosolized budesonide before OVA challenge. The levels of TGF-beta(1) in bronchoalveolar lavage fluid were analyzed by ELISA. The expressions of TGF-beta type I receptor, TGF-beta type II receptor, Smad2/3, Smad4, Smad7, and SPs were measured by immunochemistry technique and computerized image analysis system. SP-A and SP-B were significantly decreased after allergic sensitization and challenge, accompanied by active TGF-beta/Smad signal transduction. A dramatic increase in the expressions of SP-A and SP-B was observed after budesonide treatment. This was also accompanied by up-regulation of Smad7 expression and down-regulation of TGF-beta type I receptor expression. A possible explanation for the result is that an early budesonide inhaled treatment inhibits TGF-beta-induced reduction of SP-A and SP-B expression through inhibition of active TGF-beta/Smad signal transduction pathway in asthmatic mice. TGF-beta signaling may be a potentially important therapeutic target for antiasthma drugs.

Recombinant production of a hybrid plasminogen activator composed of surfactant protein B and low-molecular-weight urokinase.

Intraalveolar fibrin deposition is commonly observed during acute inflammatory and chronic interstitial lung diseases and may contribute to impairment of surfactant function and gas exchange. We recently described a chemically cross-linked chimeric protein consisting of surfactant protein (SP)-B and urokinase (uPA) for targeting alveolar fibrin under conditions such as acute respiratory distress syndrome (ARDS) or lung fibrosis. We now investigated the feasibility of a recombinant production of a fusion protein encoding mature SP-B and uPA, termed SPUC. Four different SPUC proteins (N-term SP-B/C-term uPA, N-term uPA/C-term SP-B, each +/- His-tag) were prepared by cloning the cDNA encoding mature SP-B and low-molecular-weight uPA into the expression vector pcDNA3.1. CHO-cells were transfected with the constructs and the supernatant and cell lysates were analyzed for expression of SPUC. Using a chromogenic substrate assay uPA activity was found in supernatants and lysates of transfected cells with highest activities related to the N-term uPA/C-term SP-B (+/- His-tag) construct in supernatants 48h after transfection. Casein enzymography showed an enzymatically active fusion proteins with a molecular weight of approximately 42 kDa in the supernatant of cells transfected with the N-term uPA/C-term SP-B (+/- His-tag) construct, but only a minor activity with the N-term SP-B/C-term uPA construct. The N-term uPA/C-term SP-B construct was also shown to possess higher resistance towards inhibition by plasminogen activator inhibitor-1. We conclude that recombinant production of a fusion protein consisting of mature SP-B and uPA is feasible, when the SP-B moiety is fused to the C-terminus of urokinase.

ABCA3 is critical for lamellar body biogenesis in vivo.

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3-/- knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/-, neonates survived. The lungs from E18.5 and E19.5 Abca3-/- mice were less mature than wild type. Alveolar type 2 cells from Abca3-/- embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/- compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3-/- embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.